Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting about 5 million people worldwide. The problem is expected to get worse with the aging population and currently there is no cure, vaccine, or early detection biomarkers. Extensive research on the disease has shown that microRNAs (short, non-coding RNAs) have been implicated as potential regulatory factors in the disease. Relatedly, microRNAs have been noted as promising biomarkers to detect AD or monitor its progression. Several microRNAs have already been found to have differential expression between healthy and AD individuals. Research and clinical use of microRNAs require sensitive and specific detection, which can be costly and complex using current approaches. Through a current R35 grant from NIGMS, we have developed a simple and robust assay for single-step microRNA detection. The technology is based on conformationally responsive DNA nanoswitches that loop upon recognition of a specific microRNA and report their on/off status through simple electrophoresis. Unlike many other approaches, our technique detects native microRNAs directly without labeling or amplification, eliminating the need to expensive enzymes, reagents, and equipment. This technology can be a compelling alternative for qPCR and Northern blotting and can be easily adapted by almost any researcher. In this one year supplement proposal, we will adapt this minimalistic microRNA detection assay to Alzheimer's related microRNA biomarkers in brain and blood. We will show immediate relevance to the AD research community by demonstrating quantification of a well characterized brain microRNA that is regulated in AD. We will additionally show longer term potential as a clinical tool by demonstrating multiplexed detection of a six- microRNA blood-based biomarker panel that has recently been identified. This project will establish that our programmable DNA nanoswitches can be readily adapted to detect relevant levels of microRNAs that are important to AD. Our strategy will provide two potential areas of application for our DNA nanoswitches in the context of Alzheimer's research. Successful completion of the first aim will establish its use as a viable tool for researchers in the AD field. The second aim will provide the first step toward applying the DNA nanoswitches to clinical application in detection of AD related microRNA biomarkers from bodily fluids. Individually, each of these aims will establish a foothold into different but related aspects in Alzheimer's research.